Maintaining mESC Pluripotency Flashcards
Where are mESCs derived from
E3.5 mouse = blastocyst stage, which is pre-implantation (40-80 cells)
Taken specifically from the inner cell mass, as these are the pluripotent cells
Pluripotentcy is maintained during ESC self renewal by:
- Promotion of proliferation
- Supression of differentiation through:
- Extrinsic factors (GFs, Cytokines)
- Signalling pathways
- Intrinsic factors (TFs)
mESC’s derived from d3.5 blastocystes exist in a ____ state of pluripotency because…
Naive
as they’re taken pre implantaion and gastrulation
Meaning they aren’t primed (post-gastrulation) to ecto, endo or mesoderm lineages
How was LIF (Leukaemia inhibitory factor) identifed as an extrinsic factor capable of maintaining mESC pluripotency?
mESC’s were seen to maintain PSC state when grown on feeder cells
Main factor produced by feeder cells was identified as LIF
to confirm LIF was keeping PSC state:
- gre cells with LIF + Serum
- Result = PSC state maintained without the need for feeder cells
THEREFORE LIF maintains mESCs in a pluripotent state
Why did scientists then want to study the signalling pathways activated by LIF that maintain self-renewal?
So that we can better understand self-renewal
To ultimately lead to a bigger array of molecules known to maintain PSC state in vitro
So that we can use PSC’s for therapeutic effects
How does LIF cause SR in ESCs?
- Ligand-induced dimerisation of receptor subunits
- Causes PI and activation of JAK tyrosine kinases
- These in turn PI the several tyrosine residues on the cytoplasmic domain of the gp130 receptor subunit
- These phsophotyrosine residues interact with SH2-domain containing proteins
- These proteins in turn become target for JAKs
ALL of this results in the activation of:
- RAS - MAP - ERK pathway
- JAK - STAT3 pathway
What is STAT and how does it function in ESC SR?
is a latent TF
1. Pi’d by JAK
2. Causes dimerisation
3. Can now translocate to the nucleus
4. Where theya ctivate target genes invovled in maintaining PSC state
What was unexpectedly discovered about STAT?
It causes differentiation in some somatic cells:
- Myloid leukemia M1 cells
- Neuroepitheial precursor cells that become astrocytes
What is the importance of the gp130 docking sites and STAT function?
gp130 cytoplasmic domain has 7 tyrosine residues - 4 of which are sites of STAT3 activation
- Mutate these 4 = elimination of SR signal and STAT3 DNA binding
THEREFORE, the docking dites on gp130 are important for STAT function
What are the two subunits that make the LIF receptor?
heterodimer
1. low-affinity LIF receptor (LIF)
2. gp130 signal transducer
What are some other LIF-related cytokines that act through the same receptor as LIF?
Oncostatin M
Cardiotrophin
What are some LIF-related cytokines that act through gp130 homodimers?
IL-6
(interleukin-6)
How were BMP 2 and 4 discovered to be additional factors controlling mESC PSC state? what was concluded from it? (Thomson et al, 1998)
no serum + LIF = PSC state lost
BMP2/4 + LIF = PSC state maintaines
BMP2/4 + no LIF = PSC state lost
no BMP2/4 + no LIF = PSC state lost
concluded that SR response remains dependent on continues LIF and BMP2/4 signalling
How do BMP2/4 promote SR in mESCs?
- Bind to TGF-beta type 1 and 11 serine/theronine receptors
- These heterodimerise
- PI SMAD’s 1,8,5
- These combine with SMAD-5
- Translocate to nucleus
- Promote IDs (inhibitors of differentiation)
- Causes transcription of genes promoting SR
As both BMPs and LIF are required to maintain PSC state of mESC’s, do their signalling cascades cross at all?
Yes - in some places
1. SMAD and STAT3 form a ternary complex with P300 that results in differentiation
2. BMP, no LIF activates some LIF specific gene targets, ones that promote SR not diff
